Project Summary Airway submucosal glands (SMGs) are thought to contain facultative stem cell niches for the surface airway epithelium (SAE). In mice, this niche serves only the trachea; however, in larger mammals such as humans, pigs and ferrets, SMGs are present throughout the cartilaginous airways and may serve the broader function of maintaining the proximal conducting airway epithelium in the setting of disease. In cystic fibrosis (CF), defects in human, pig, ferret, and mouse CFTR-mediated SMG secretions lead to alterations in the expression of the neuroendocrine peptide CGRP, which promotes airway progenitor cell proliferation following injury. In CF mice, this pathology alters the characteristics of slowly-cycling stem cells in both the SMG and SAE niches, suggesting that glandular dysfunction in CF may impact mechanisms involved in airway repair. During the previous five funding cycles, this grant has addressed multiple aspects of airway SMG biology, SMG stem/progenitor cells, and CF pathogenesis. Recently, our studies have focused on elucidating the Wnt- dependent mechanisms that overlap in the control of SMG development and the adult SMG stem cell niche. These studies have shown that Wnt-dependent changes in Lef-1 and Sox2 expression are key to the commitment of primordial glandular stem cells to form SMGs. We now have evidence that Wnt-active niches within adult SMGs may play an important role in regulating slowly-cycling stem cells and that conditional deletion or overexpression of Lef-1 influences glandular stem cell properties. Using lineage tracing, we have demonstrated that myoepithelial cells of SMGs can differentiate into both glandular and SAE cell types. We propose to use lineage tracing in Lef-1 and Sox2 conditional knockout and overexpressing mice to investigate how these two transcription factors control the Wnt pathways that are critical for mobilizing SMG stem cells following airway injury. Using a BATgal Wnt-reporter transgenic line, we have demonstrated that this reporter specifically marks primordial glandular stem cells, glandular niches in adult SMGs associated with resident slowly-cycling stem cells, and the first cycling stem/progenitors within SMGs following injury. Using CRISPR/Cas9-mediated gene editing in BATgal zygotes, we have converted this transgenic reporter line into a BATCreERT2 driver mouse, which will enable lineage-tracing and conditional gene deletion studies of this unique Wnt-regulated compartment. Lastly, we will dissect how SMG defects in CF mice impact the stem cells that reside in the glandular niche using lineage-tracing of glandular myoepithelial cells and Wnt-activated progenitors following airway injury. This project will enhance our understanding of stem cell phenotypes in airway SMGs, which have the capacity to generate both glandular and SAE cell types. Furthermore, this work will delineate the disease-associated changes to SMG stem cell niches that may be important for the pathogenesis of CF airway disease and other hypersecretory diseases affecting SMGs such as asthma and chronic bronchitis.